Use of heterocyclic inhibitors of serine proteases

ABSTRACT

Certain novel heterocyclic compounds, their preparation, and their use in inhibiting serine proteases with chymotrypsin-like, elastase-like, and trypsin-like specificity and in the treatment of diseases such as emphysema which involve tissue proteolysis.

This application is a continuation-in-part of Ser. No. 07/215,994, filed Jul. 7, 1988, now abandonned, which is a continuation of Ser. No. 07/874,459, filed Jun. 13, 1986, now abandonned, which is a continuation of Ser. No. 06/642,995, filed Aug. 20, 1984, now U.S. Pat. No. 4,596,822.

BACKGROUND OF THE INVENTION

This invention relates to a novel class of heterocyclic compounds useful for selectively inhibiting elastase, selectively inhibiting chymotrypsin-like enzymes, selectively inhibiting trypsin-like enzymes, or for generally inhibiting serine proteases of all classes. Certain clinical symptoms found in pancreatitis, emphysema, rheumatoid arthritis, inflammation, and adult respiratory distress syndrome are believed to be caused by uncontrolled elastase in the affected tissues. Likewise, similar clinical symptoms found in the same diseases are believed to be caused by uncontrolled cathepsin G, mast cell chymase, and other chymotrypsin-like enzymes. In vitro, proteolysis by serine proteases of the elastase, chymotrypsin, and trypsin families is often a severe problem in the production, isolation, purification, transport and storage of valuable peptides and proteins.

It is an object of this invention to find a novel group of specific inhibitors for elastase, chymotrypsin, trypsin, and other serine proteases of similar substrate specificity, and serine proteases in general. Inhibitors are substances which reduce or eliminate the catalytic activity of enzymes. Chymotrypsin and chymotrypsin-like enzymes normally cleave peptide bonds in proteins and peptides where the amino acid residue on the carbonyl side of the split bond (P₁ residue) is typically Trp, Tyr, Phe, Met, Leu or other amino acid residues which contain aromatic or large alkyl side chains. Elastase and elastase-like enzymes, on the other hand, cleave peptide bonds where the P₁ amino acid residue is much smaller, typically Ala, Val, Ser, Leu and other similar amino acids. Trypsin-like enzymes hydrolyze peptide bonds where the P₁ amino acid is Lys or Arg. All of the above enzymes have extensive secondary specificity and recognize amino acid residues removed from the P₁ residue. The inhibitors of this invention would be useful for treating diseases such as pancreatitis, emphysema, rheumatoid arthritis, adult respiratory distress syndrome, and inflammatory diseases which involve destruction of tissue by serine proteases. In some cases, it would be more useful to utilize a specific elastase, trypsin or chymotrypsin-like enzyme inhibitor, while in other cases an inhibitor with broader specificity would be appropriate.

It is an object of this invention to find a novel group of specific inhibitors useful in vitro for inhibiting elastase, trypsin, chymotrypsin and other serine proteases of similar specificity, and for inhibiting serine proteases in general. Such inhibitors could be used to identify new proteolytic enzymes encountered in research. They could also be used in research and industrially to prevent undesired proteolysis that occurs during the production, isolation, purification, transport and storage of valuable peptides and proteins. Such proteolysis often destroys or alters the activity and/or function of the peptides and proteins. Uses would include the addition of the inhibitors to antibodies, enzymes, plasma proteins, tissue extracts, or other proteins which are widely sold for use in clinical analysis, biomedical research, and for many other reasons. For some uses a specific inhibitor would be desirable, while in other cases, an inhibitor with general specificity would be preferred.

DETAILED DESCRIPTION OF THE INVENTION

Certain substituted isocoumarins have been found to be excellent inhibitors of a number of serine proteases including human leukocyte elastase, porcine pancreatic elastase, bovine chymotrypsin, human cathepsin G, various human and bovine blood coagulation enzymes, human complement factor D, and several mammalian mast cell proteases. These compounds inhibit the serine proteases by acylating the active site serine residue and in some cases form an additional covalent bond. These structures may be used in vivo to treat diseases resulting from tissue destruction due to elastase, chymotrypsin, trypsin, and related enzymes. They may be used in vitro to prevent proteolysis that occurs during the production, isolation, purification, storage, and transport of peptides and proteins. The novel substituted isocoumarins and related heterocyclic analogs have the following structural formula: ##STR1## wherein

X is selected from the group consisting of O and S,

Z is selected from the group consisting of H, halogen, C₁₋₆ alkyl, C₁₋₆ alkyl with an attached phenyl, C₁₋₆ fluorinated alkyl, C₁₋₆ alkoxy, C₁₋₆ fluorinated alkoxy, C₁₋₆ alkoxy with an attached phenyl, benzyloxy, 4-fluorobenzyloxy, --OCH₂ C₆ H₄ --R' (2-substituent), --OCH₂ C₆ H₄ --R' (3-substituent), --OCH₂ C₆ H₄ -R' (4-substituent), --OCH₂ C₆ H₃ -R₂ ' (2,3-substituents), --OCH₂ C₆ H₃ -R₂ ' (2,4-substituents), --OCH₂ C₆ H₃ -R₂ ' (2,5-substituents), --OCH₂ C₆ H₃ -R₂ ' (2,6-substituents), --OCH₂ C₆ H₃ -R₂ ' (3,4-substituents), and --OCH₂ C₆ H₃ -R₂ ' (3,5-substituents),

R' is selected from the group consisting of H, halogen, trifluoromethyl, NO₂, cyano, methyl, methoxy, acetyl, carboxyl, OH, and amino,

Y is selected from the group consisting of H, halogen, trifluoromethyl, methyl, OH, and methoxy,

R is selected from the group consisting of H, OH, NH₂, NO₂, halogen, --NH--C(NH)--NH₂, --C(NH)NH₂, C₁₋₆ alkoxy, C₁₋₆ fluorinated alkoxy, C₁₋₆ alkyl, C₁₋₆ alkylamino, M-AA, and M-NH,

wherein AA is selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-amincaproic acid, citrulline, hydroxyproline, ornithine, and sarcosine,

wherein M is selected from the group consisting of hydrogen, lower alkanoyl having 1 to 6 carbons, carboxylalkanoyl, hydroxyalkanoyl, amino-alkanoyl, benzene sulfonyl, tosyl, benzoyl, and lower alkyl sulfonyl having 1 to 6 carbons.

Several of these compounds have been prepared earlier for other purposes (illustrative examples: Davies and Poole, J. Chem. Soc., pp 1616-1629, (1928); Milevskaya, Belinskaya, and Yagupol' skii, Zhur. Org. Khim. 9, pp 2145-2149, (1973); Tirodkar and Usgaonkar, Ind. J. Chem. 7, pp 1114-1116, (1969); Choksey and Usgaonkar, Ind. J. Chem. 14B, pp 596-598, (1976)).

The following compounds are representative of the invention:

3-chloroisocoumarin,

3,4-dichloroisocoumarin,

3-ethoxy-4-chloroisocoumarin,

3-isobutyloxy-4-chloroisocoumarin,

3-benzyloxy-4-chloroisocoumarin,

3-(4-fluorobenzyloxy)-4-chloroisocoumarin,

7-amino-3-methoxy-4-chloroisocoumarin,

7-amino-3-ethoxy-4-chloroisocoumarin,

7-amino-3-benzyloxy-4-chloroisocoumarin,

7-nitro-3-methoxy-4-chloroisocoumarin,

7-nitro-3-ethoxy-4-chloroisocoumarin,

7-nitro-3-benzyloxy-4-chloroisocoumarin,

7-hydroxy-3-ethoxy-4-chloroisocoumarin,

7-nitro-3-methoxyisocoumarin,

7-nitro-3-(2-phenethoxy)-4-chloroisocoumarin,

7-amino-3-(2-phenethoxy)-4-chloroisocoumarin, and

7-(N-tosyl-phenylalanylamino)-4-chloro-3-methoxyisocoumarin.

7-Acetylamino-4-chloro-3-methoxyisocoumarin

7-Acetylamino-4-chloro-3-ethoxyisocoumarin

7-Acetylamino-4-chloro-3-propyloxyisocoumarin

7-[(N-Tosyl-alanyl)amino]-4-chloro-3-methoxyisocoumarin

7-[(N-Tosyl-valyl)amino]-4-chloro-3-methoxyisocoumarin

7-([N-Tosyl-leucyl)amino]-4-chloro-3-methoxyisocoumarin

7-[(N-Tosyl-phenylalanyl)amino]-4-chloro-3-methoxyisocoumarin

7-[(N-Tosyl-phenylalanyl)amino]-4-chloro-3-propyloxyisocoumarin

7-Butanoylamino-4-chloro-3-propyloxyisocoumarin

7-Isovaleroylamino-4-chloro-3-propyloxyisocoumarin.

When R is H, NH₂, NO₂, X is O, and Y and Z are any of the noted groups, the isocoumarin structure is a general inhibitor for both human leukocyte (HL) elastase and bovine chymotrypsin. Although these substituted isocoumarins are slightly less effective toward porcine pancreatic (PP) elastase and cathepsin G, they are still capable of inhibiting these enzymes. The rate constants for inactivation of HL elastase, PP elastase, and chymotrypsin by 3-chloroisocoumarin (X=O, Z=Cl, and Y=H) have been measured and are published, (Harper, Hemmi, and Powers, J. Amer. Chem. Soc. 105, pp 6518-6520 (1983)). This publication is incorporated herein by reference. The 3-chloroisocoumarin also inactivates rat mast cell proteases I and II and Streptomyces griseus protease A with rates of 84, 85, and 196 M⁻¹ s⁻¹, respectively. Rate constants for inactivation of a number of elastase, chymotrypsin, and trypsin-like enzymes by 3,4-dichloroisocoumarin (R=H, X=O, Y=Z=Cl) are given in Table I. This table indicates the generality of this compound as a serine protease inhibitor.

                  TABLE I                                                          ______________________________________                                         Inactivation of Proteases by 3,4-Dichloroisocoumarin.sup.a.                                  Inhibitor                                                                      Concentration                                                                              t.sub.1/2 k.sub.obsd /[I]                            Enzyme        (M)         (min)     (M.sup.-1 s.sup.-1)                        ______________________________________                                         Human Leukocyte                                                                              1.1         1.2       8920                                       Elastase.sup.b                                                                 Porcine Pancreatic                                                                           8.1         0.57      2500                                       Elastase                                                                       Human Leukocyte                                                                              49.0        8.4       28                                         Cathepsin G                                                                    Rat Mast Cell 38.0        1.2       259                                        Protease I                                                                     Rat Mast Cell 11.0        1.8       583                                        Protease II.sup.b                                                              Human Skin Chymase                                                                           92.0        4.7       27                                         Bovine Chymotrypsin-A                                                                        13.0        1.6       566                                        S. griseus Protease A                                                                        136.0       0.3       306                                        Subtilisin                          substrate                                  Human Thrombin.sup.c                                                                         340.0       3.4       10                                         Bovine Thrombin                                                                              127.0       3.7       25                                         Human Plasmin 203.0       0.4       133                                        Porcine Pancreatic                                                                           127.0       3.4       27                                         Kallikrien.sup.c                                                               Bovine Factor Xa.sup.c                                                                       422.0       133.0     0.2                                        Bovine factor XIa.sup.c                                                                      239.0       1.8       27                                         Human Factor XIIa.sup.c                                                                      135.0       1.3       64                                         Human Factor D                                                                               109.0       0.6       192                                        Bovine Trypsin                                                                               127.0       0.5       198                                        S. aureus Protease V-8.sup.e                                                                 18.0        0.3       2765                                       Acetylcholinesterase.sup.f                                                                   157.0       >120      <0.6                                       Beta-Lactamase.sup.f                                                                         385.0                 .sup. NI.sup.g                             Papain.sup.h  422.0                 NI                                         Leucine aminopeptidase.sup.e                                                                 400.0                 NI                                         ______________________________________                                          .sup.a Inactivation measurements were performed using the incubation           method in 0.1M Hepes, 0.5M NaCl, 8-10% Me.sub.2 SO, pH 7.5 using the           incubation method. An aliquot of inhibitor was added to an enzyme solutio      and aliquots removed with time and assayed for remaining enzymatic             activity. First order rate constants, k.sub.obsd, were obtained from the       slope of plots of ln (v/v.sub.o) versus times.                                 .sup.b Data obtained by progress curve method of Tian and Tsou,                Biochemistry 21, 1028-1032, (1982).                                            .sup.c Incubation and assay buffer was 0.1M Hepes, 5 mM CaCl.sub.2, 8-10%      Me.sub.2 SO, pH 7.5.                                                           .sup.d Data obtained using the progress curve method (0.1M Hepes, 5 mM         CaCl.sub.2, 10% Me.sub.2 SO, pH 7.5).                                          .sup.e Buffer was 0.1M Hepes, 10% Me.sub.2 SO, pH 7.5.                         .sup.f Buffer was 0.1M phosphate, 10% Me.sub.2 SO, pH 7.0.                     .sup.g NI, no inactivation.                                                    .sup.h Buffer was 0.05M Tris HCl, 5 mM cysteine, 2 mM                          ethylenediaminetetraacetic acid, 10% Me.sub.2 SO, pH 8.2.                

Table II shows the inactivation rate constants for several serine proteases inhibited by substituted 3-alkoxyisocoumarins. These k_(obsd) /[I] values are second order inactivation rate constants and reflect irreversible inactivation of the enzyme. In some cases (see Tables I and II), inactivation rate constants were measured in the presence of substrate using the progress curve method as described by Tian and Tsou, Biochemistry 21, pp 1028-1032, (1982). Significant, and in most cases total, inactivation of the enzyme will occur if the inhibitor concentration is chosen to be 5 to 50 times the enzyme concentration. The 7-amino-4-chloro-3-methoxyisocoumarin is an essentially stoichiometric inactivator of PP elastase and chymotrypsin.

The rate at which the enzyme is inactivated can be altered by changing both R, Z, and Y. The structures with long alkoxy or benzyloxy substituents are best at inhibiting chymotrypsin, while the most effective HL elastase inhibitors contain Z groups of Cl and C₁₋₄ alkyloxy. Isocoumarin itself (X=O, Z=Y=H, and R=H) does not inhibit serine proteases. One of the most effective inhibitors of the blood coagulation proteases and other trypsin-like enzymes was 3,4-dichloroisocoumarin (Z=Y=Cl, R=H, X=O). Attachment of positively charged groups such as guanidine or amines in R or Z would make the inhibitor reactive towards trypsin-like enzymes. Additional specificity towards a particular protease could be achieved by the placing the proper aminoacid or peptide derivative on the 7-amino group. Thus the specificity or generality of the inhibition reaction can be controlled first, by choosing the appropriate inhibitor structure and second, by choosing the inhibitor concentration utilized.

                                      TABLE II                                     __________________________________________________________________________     Rate Constants (k.sub.obsd /[I]) for Inactivation of Serine Proteases          by Substituted-4-chloroisocoumarins.sup.a.                                                Enzyme                                                              Inhibitor  HLE.sup.b                                                                           PPE.sup.c                                                                          Cat G.sup.d                                                                        Chyl.sup.e                                                                         RMCP II.sup.f                                                                        SGPA.sup.g                                   __________________________________________________________________________     3-ethoxy-4-chloro-                                                                        .sup. 43000.sup.h                                                                   940 190  610                                                                               3050   820                                         isocoumarin                                                                    3-isobutyloxy-                                                                             9500                                                                                NI.sup.j                                                                          190  750                                                                                920  6400                                         4-chloroisocoumarin                                                            3-benzyloxy-                                                                               1525                                                                                6  1140                                                                               .sup. 16000.sup.k                                                                  9600  5500                                         4-chloroisocoumarin                                                            3-(4-fluorobenzyloxy-                                                                      2300                                                                               NI  216  32000.sup.l                                                                       3200  3350                                         4-chloroisocoumarin                                                            7-nitro-3-methoxy-                                                                         2580                                                                               580                                                            4-chloroisocoumarin                                                            7-nitro-3-ethoxy-                                                                          2800                                                                               1300                                                                               NI   4300                                                                              7000  2700                                         4-chloroisocoumarin                                                            7-nitro-3-benzyloxy-                                                                               2600                                                                               10500                                                                              10500                                              4-chloroisocoumarin                                                            7-amino-3-methoxy-                                                                        14000                                                                               1035                                                                                17  108                                                                                130   40                                          4-chloroisocoumarin                                                            7-amino-3-ethoxy-                                                                          9420                                                                               700 195  274                                                                                876   216                                         4-chloroisocoumarin                                                            7-(Tos-Phe-NH)-                                                                           190000                                                                              6480                                                                               195  274                                                                               NI    NI                                           4-chloro-3-methoxy-                                                            isocoumarin                                                                    __________________________________________________________________________      .sup.a Conditions were 0.1M Hepes, 0.5M NaCl, pH 7.5, 10% Me.sub.2 SO, at      25° C. Rate constants were obtained by the incubation method unles      otherwise noted. An aliquot of inhibitor was added to a solution of enzym      and aliquots removed with time and assayed for remaining enzymatic             activity. The first order rate constants, k.sub.obsd, were obtained from       plots of ln (v/v.sub.o) versus times. The units of k.sub.obsd /[I] are         M.sup.-1 s.sup.-1.                                                             .sup.b Inhibitor concentrations were from 0.007 to 0.011 mM.                   .sup.c Inhibitor concentrations were from 0.037 to 0.011 mM.                   .sup.d Inhibitor concentrations were from 0.113 to 0.009 mM.                   .sup.e Inhibitor concentrations were from 0.013 to 0.098 mM.                   .sup.f Inhibitor concentrations were from 0.030 to 0.009 mM.                   .sup.g Inhibitor concentrations were from 0.086 to 0.005 mM.                   .sup.h Progress curve method with [I] = 175-600 nM.                            .sup.i Progress curve method with [I] = 0.012 mM.                              .sup.j NI, no inactivation.                                                    .sup.k Progress curve method with [I] = 0.0003-0.0017 mM.                      .sup.l Progress curve method with [I] = 0.0012 mM.                       

The spontaneous hydrolysis rates of many of these substituted isocoumarins in buffer solution have been measured and are summarized in Table III. The hydrolysis rates are dependent upon the composition of the buffer. In general, these isocoumarins are more stable in phosphate buffered saline (pH 7.4) than in Hepes buffer (pH 7.5). The most stable inhibitor studied was 7-amino-3-methoxy-4-chloroisocoumarin while the most unstable was 3,4-dichloroisocoumarin. In addition, 7-amino-3-methoxy-4-chloroisocoumarin is quite stable in albumin (0.4 mg/ml) while 3,4-dichloroisocoumarin decomposed rapidly upon addition to albumin (0.4 mg/ml). The 3-alkoxy-4-chloroisocoumarins are intermediate in stability. These compounds are significantly more stable than other serine protease inhibitors such as aza-peptides and sulfonyl fluorides.

                  TABLE III                                                        ______________________________________                                         Half-lives for Spontaneous Hydrolysis of Substituted                           Isocoumarins in Buffer Solution.sup.a.                                                        t.sub.1/2 (min)                                                                  Hepes   Phosphate                                             Compound         pH 7.5.sup.b                                                                           pH 7.4.sup.c                                          ______________________________________                                         3-chloroisocoumarin                                                                             140     360                                                   3,4-dichloroisocoumarin                                                                         18       48                                                   3-ethoxy-4-chloro-                                                                              144                                                           isocoumarin                                                                    3-benzyloxy-4-chloro-                                                                           68                                                            isocoumarin                                                                    3-(4-fluorobenzyloxy)-                                                                          53                                                            4-chloroisocoumarin                                                            7-amino-3-methoxy-                                                                              195     820                                                   4-chloroisocoumarin                                                            ______________________________________                                          .sup.a Spontaneous hydrolysis rates were measured spectrophotometrically       by monitoring the decrease in absorbance due to the isocoumarin ring           system (wavelength range: 385-325 nm) and these rates converted to             halflives using the first order rate law.                                      .sup.b Conditions were: 0.1M Hepes buffer, 0.5M NaCl, 10% Me.sub.2 SO at       25° C.                                                                  .sup.c Conditions were: 0.02M K.sub.2 HPO.sub.4, 0.15M NaCl, 10% Me.sub.2      SO at 25° C.                                                      

It has been found that certain heterocyclic compounds will inhibit serine proteases such as elastase and chymotrypsin by blocking the active site of the enzyme. The compounds of this invention are 3-substituted-7-substituted-(1H)2-benzopyran-1,4(3H)-diones. The unsubstituted (1H)2-benzopyran-1,4(3H)-dione has been prepared previously for other purposes (cf. Knott, J. Chem. Soc., pp 402-410 (1963). The heterocyclic serine protease inhibitors of this invention have the following structural formula: ##STR2## wherein

R₁ is selected from the group consisting of H, halogen, C₁₋₆ alkyl, C₁₋₆ alkyl with an attached phenyl, C₁₋₆ fluorinated alkyl, C₁₋₆ alkoxy, C₁₋₆ fluorinated alkoxy, C₁₋₆ alkoxy with an attached phenyl, benzyloxy, 4-fluorobenzyloxy, --OCH₂ C₆ H₄ --R' (2-substituent), --OCH₂ C₆ H₄ --R' (3-substituent), --OCH₂ C₆ H₄ --R' (4-substituent), --OCH₂ C₆ H₃ --R₂ ' (2,3-substituents), --OCH₂ C₆ H₃ --R₂ ' (2,4-substituents), --OCH₂ C₆ H₃ --R₂ ' (2,5-substituents), --OCH₂ C₆ H₃ --R₂ ' (2,6-substituents), --OCH₂ C₆ H₃ --R₂ ' (3,4-substituents), and --OCH₂ C₆ H₃ --R₂ ' (3,5-substituents),

R' is selected from the group consisting of H, halogen, trifluoromethyl, NO₂, cyano, methyl, methoxy, acetyl, carboxyl, OH, and amino,

R₂ is selected from the group consisting of H, OH, NH₂, NO₂, halogen, --NH--C(NH)--NH₂, --C(NH)NH₂, C₁₋₆ alkoxy, C₁₋₆ fluorinated alkoxy, C₁₋₆ alkyl, and C₁₋₆ alkylamino.

The following novel compounds are representative of this invention:

(1H)2-benzopyran-1,4(3H)-dione,

3-ethyl-(1H)2-benzopyran-1,4(3H)-dione,

3-propyl-(1H)2-benzopyran-1,4(3H)-dione,

3-phenyl-(1H)2-benzopyran-1,4(3H)-dione.

These structures inhibit serine proteases by forming relatively stable acyl enzymes. HL elastase (0.0004 mM) is totally inactivated by 3-propyl-1H-2-benzopyran-1,4(3H)-dione (0.143 mM) while chymotrypsin (0.002 mM) is totally inhibited by this compound at a concentration of 0.2 mM. The unsubstituted 1H-2-benzopyran-1,4(3H)-dione is a less effective inhibitor than the propyl derivative towards HL elastase and chymotrypsin. These compounds are more potent inhibitors of HL elastase and chymotrypsin than cathepsin G and PP elastase. As with the substituted isocoumarins mentioned earlier, the specificity of these structures towards a particular serine protease can be partially controlled by the nature of the substituent groups (R₁ and R₂).

In the compounds of the present invention, the R group is critical for effective inhibition, and, no compounds currently are known in the art with one of the listed R groups. There also are no compounds currently known in the literature or the prior art that are remotely related to the compounds claimed in the present invention. The few known compounds have R═H, which is not claimed in the present invention.

To use the above identified inhibitors in vitro, they are dissolved in an organic solvent such as dimethylsulfoxide or ethanol and are added to an aqueous solution containing the protease which is to be inhibited such that the final concentration of organic solvent is 25% or less. The inhibitors may also be added as solids or in suspension.

The amounts of active ingredients, suitable carriers, and modes of administration to employ to treat emphysema are well-known. The paper by Powers, J. C., Am. Rev. Respir. Dis. 127, s54-s58 (1983), which is incorporated herein by this reference, cites examples of use of elastase inhibitors to treat emphysema in animals in which the inhibitors were given orally, by injection or by instillation in the lungs and various carriers were utilized. The appropriate references are 32, 33, 34, 35, 36, 37, 38 and 39 referenced at the end of the above paper. The Applicant is a coauthor on three of these papers.

Additionally, a number of patents teach how to administrate elastase inhibitors in animals. They include thienamycin derivatives as anti-inflammatory agents, U.S. Pat. No. 4,465,687; 1-carbapenem-3-carboxylic esters as anti-inflammatory agents, U.S. Pat. No. 4,493,839; and halogenated protease inhibitors, U.S. Pat. No. 4,469,885.

The serine protease inhibitors of this invention would be useful in a variety of experimental procedures where proteolysis is a significant problem. Inclusion of these inhibitors in radioimmunoassay experiments would result in higher sensitivity. The use of these inhibitors in plasma fractionation procedures would result in higher yields of valuable plasma proteins and would make purification of the proteins easier. The inhibitors disclosed here could be used in cloning experiments utilizing bacterial cultures, yeast and E. coli and would result in a more easily purified cloned product in higher yield.

There is conclusive evidence that the compounds of the present invention function in the same way as the beta-lactam inhibitors disclosed in U.S. Pat. Nos. 4,465,687 and 4,493,839 (the '687 and '839 patents, respectively). The thienamycin derivatives and 1-carbapenem-3-carboxylic esters in these two patents are beta-lactam derivatives. The similarity between the compounds of the present invention and the beta-lactam of the above two patents are:

1. The present compounds and the beta-lactam derivatives both inhibit elastase which is the enzyme which digests the lungs and causes emphysema. The table on pages 8, 9, and 10 of U.S. Pat. No. 4,493,839 and on page 7 of U.S. Pat. No. 4,465,687 shows the elastase inhibitor potency of the beta-lactam inhibitors. The inhibitor potency of the beta-lactam inhibitors is also published. The inhibitory potency of the present compounds is given in the specification and in the papers incorporated therein by reference.

2. Both the present compounds and the beta-lactam inhibitors disclosed in the '687 and '839 patents have the same degree of elastase inhibitory acitivity.

3. Both the present compounds and the beta-lactam inhibitors disclosed in the '687 and '839 patents are irreversible inhibitors of elastase. 4. Both the present compounds and the beta-lactam inhibitors disclosed in the '687 and '839 patents inhibit elastase by forming a covalent ester bond with the active site serine-195 residue of the enzyme.

5. In addition, some of the present compounds also inhibit elastase by forming an additional covalent bond with the active site histidine-57 of elastase. Some of the beta-lactam inhibitors disclosed in the '687 and '839 patents also have this property.

For the present compounds, see Dogherty, R. M., et al. (1986) Nature 322, 192-194, Bode, W. et al., (1989) Biochemistry, 28, 1951-1963, Meyer, E. F., L. G. Presta and R. Radhakrishnan (1985) J. Am. Chem. Soc. 107, 4091-4093, and Navia, N. A., et al. (1987) Nature 327, 79-82, which are incorporated herein by this reference.

The amounts of active ingredients, suitable carriers, and modes of administration suitable for use with the present invention to employ and to treat emphysema are known in the art. The following discussion is applicable to the present invention as the amounts, carriers and modes of administration for elastase inhibitors are interrelated, as known by those skilled in the art.

Several different types of synthetic elastase inhibitors have been used in animal studies of emphysema. These have included peptide chloromethyl ketones, furyl saccharine, and trifluoroaceteyldipepetides. However, peptide chloromethyl ketones have been the most widely studied.

The chloromethyl ketone Ac-Ala-Ala-Pro-AlaCH₂ Cl has been extensively studied in hamsters. Experimental emphysema was induced by intratracheal instillation of porcine pancreatic elastase. The inhibitor was administered intraperitoneally in divided doses before and immediately after instillation of the elastase. The inhibitor was shown to have significant antielastase activity in vivo and markedly decreased the extent of elastase-induced emphysema. In addition, it was shown that the development of emphysema was dependent on the dose of Ac-Ala-Ala-Pro-AlaCH₂ Cl with emphysema being eliminated with 4.1 and 8.0 mg doses and markedly diminished with 1.1 mg.

The chloromethyl ketone Ac-Ala-Ala-Pro-AlaCH₂ Cl was not effective when administered 60 min after exposure to elastase. A divided dose of 19 mg was injected intraperitoneally. In addition, this large dose was shown to produce a unique renal tubular nephropathy. In the previous studies when the inhibitor was administered concurrently with the elastase, no evidence of toxicity or kidney injury was observed with a similar dose. The mechanism of this in vivo toxicity is not yet known.

The chloromethyl ketone MeO-Suc-Ala-Ala-Pro-ValCH₂ Cl has been shown to be orally active in protecting Swiss-Webster mice from elastase-induced emphysema. The mice were given a transoral dose of porcine pancreatic elastase and were given 300 to 400 mg of the chloromethyl ketone at times ranging from 2 h before instillation of the enzyme to 48 h after the enzyme. Mice that received the elastase inhibitor within 15 min before instillation of elastase were protected from development of emphysema and their lungs were completely free of alveolar deformation. However, no significant protection was afforded by treatment with MeO-Suc-Ala-Ala-Pro-ValCH₂ Cl 1 h or more before the challenge with the enzyme or at any time after the challenge.

The chloromethyl ketone Suc-Ala-Ala-Pro-ValCH₂ Cl has been shown by morphometric and physiologic measurements to moderate elastase-induced emphysema in hamsters. When 0.5 mg of the inhibitor in saline was injected intratracheally 1 h before intratracheal injection of porcine pancreatic elastase, the hamsters did not develope emphysema. When the inhibitor was injected 1 h after the elastase, the severity of emphysema was reduced by approximately 50% compared with control animals receiving saline 1 hr after elastase. The Suc-Ala-Ala-Pro-ValCH₂ Cl was ineffective when administered intratracheally 4 h after the elastase.

To explore the reasons for the ineffectiveness of peptide chloromethyl ketones when administered after the dose of elastase, the reaction of elastase with substrate and inhibitors in the presence of elastin was examined. When HL elastase or porcine pancreatic elastase is added to excess elastin, the ability of the enzyme to hydrolyze a small synthetic substrate is only slightly diminished. This indicates that the elastase molecules bound to elastin are slightly less available to the substrate or that a slight alteration in the active site conformation has occurred. In addition, binding of the elastase to the elastin is complete within a few minutes.

Rates of inhibition of elastase by peptide chloromethyl ketones were then measured in the presence and absence of elastin. In all cases, the chloromethyl ketone elastase inhibitors examined were much less effective at inhibiting elastase preabsorbed onto elastin. Indeed, with the chloromethyl ketones used in the animal emphysema models (porcine pancreatic elastase induced), inhibition rates were decreased by two-to eightfold. Even larger rate reductions were observed with HL elastase preabsorbed onto elastin. These low rates of inhibition may explain the ineffectiveness of peptide chloromethyl ketone elastase inhibitors in the animal models when administered after the dose of elastase. After the elastase has had an opportunity to bind to lung elastin, it is much harder to inhibit.

Only a few other classes of elastase inhibitors have been studied in animal models. Using the papain-induced emphysema model in hamsters, chaulmoogric acid, furoyl saccharin, the azapeptide Ac-Ala-Ala-Pro-NHN(CH₃)CO-OCH(CH₃)CONH₂, and elastatinal have been shown to be effective inhibitors. The dipeptide CF₃ CO-Lys-Ala-NHC₆ H₅ and the chloromethyl ketone CF₃ CO-AlaCH₂ Cl have been tested in the porcine pancreatic elastase-induced emphysema model in hamsters. The chloromethyl ketone gave significant partial protection against the emphysematous lesions, but no protection was obtained with the dipeptide. This is an excellent in vitro elastase inhibitor and possibly it is cleaved at the Lys residue in vivo by some trypsin-like enzyme. Interestingly, it has been reported in an abstract that a_(I) -protease inhibitor or elastatinal were partially effective at suppressing emphysematous lesions in the hamster-elastase model when the inhibitors were administered 6 days after the elastase instillation. This observation should be confirmed independently by other investigators because other types of inhibitors (peptide chloromethyl ketones) have been shown to be ineffective at such a long interval after elastase instillation.

For treatment of inflammation, fever or pain, the compounds of Formula (I) disclosed in U.S. Pat. No. 4,493,839 may be administered orally, topically, parenterally, by inhalation spray or rectally in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants and vehicles. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection or infusion techniques. In addition to the treatment of warm-blooded animals such as mice, rats, horses, dogs, cats, etc., the compounds of the invention are effective in the treatment of humans.

The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents selected from the group consisting of sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide pharmaceutically elegant and palatable preparation. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be for example, inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example, maize starch, or alginic acid; binding agents, for example starch, gelatin or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. The tablets may be uncoated or they may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time delay material such as glyceryl monsterate or glyceryl disterate may be employed.

Formulations for oral use may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example peanut oil, liquid paraffin, or olive oil.

Recent studies indicate that esterification of the carboxyl group b-lactam antibiotics can improve significantly the oral bioavailability of the parent drug. See W. E. Wright et al., The Journal of Antibiotics, XXXII, No. 11, 1155 (1979). Accordingly, esters of the compounds of formula (1) disclosed in U.S. Pat. No. 4,493,839 which can be easily hydrolyzed to acids of formula (I) disclosed in U.S. Pat. No. 4,493,839 such as acetoxymethyl ester, (5-methyl or alkyl-2-oxo-1,3-dioxolen-4-yl)unethyl ester or methoxymethyl ester of compound (I) disclosed in U.S. Pat. No. 4,493,839 are prepared for oral formulation. For example, oral solutions or suspensions may be prepared from an acetoxymethyl ester of compound (I) disclosed in U.S. Pat. No. 4,493,839 in saline or 50% aqueous propylene glycol.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipient are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents may be a naturally-occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol, or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyoxyethylene sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate. The said aqueous suspensions may also contain one or more preservatives, for example ethyl, or n-propyl, p-hydroxybenzoate, one or more coloring agents, one or more flavoring agents, and one or more sweetening agents, such as sucrose or saccharin.

Oily suspension may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oily suspensions may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of any antioxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent and one or more preservatives. Suitable dispersing or wetting agents and suspending agents are exemplified by those already mentioned above. Additional excipients, for example sweetening, flavoring and coloring agents, may also be present.

The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oils, or a mineral oil, for example liquid paraffin or mixtures of these. Suitable emulsifying agents may be naturally-occurring gums, for example gum acacia or gum tragacanth, naturally-occurring phosphatides, for example soy bean, lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan mono-oleate, and condensation products of the said partial esters with ethylene oxide, for example polyoxyethylene sorbitan monooleate. The emulsions may also contain sweetening and flavoring agents.

Syrups and elixirs may be formulated with sweetening agents, for example glycerol, sobital or sucrose. Such formulations may also contain a demulcent, a preservative and flavoring and coloring agents. The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleagenous suspension. This suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The compounds of Formula (I) disclosed in U.S. Pat. No. 4,493,839 may also be administered in the form of suppositories for rectal administration of the drug. These compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials are cocoa butter and polyethylene glycols.

For topical use, creams, ointments, jellies, solutions or suspensions, etc., containing the anti-inflammatory agents are employed.

Dosage levels of the order to 0.2 mg to 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (10 mg to 7 gms. per patient per day). Preferably, the dosage is from about 0.5 to 50 mg of the compound per kilogram of body weight per day (25 mg to 3.5 gms per patient per day). Advantageously, from about 2 mg to about 20 mg per kilogram of body weight per daily dosage produces highly effective results (50 mg to 1 gm per patient per day).

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. For example, a formulation intended for the oral administration of humans may contain from 5 mg to 5 gm of active agent compounded with an appropriate and convenient amount of carrier material which may vary from about 5 to about 95 percent of the total composition. Dosage unit forms will generally contain between from about 25 mg to about 500 mg of active ingredient.

It is well known in the literature that in vitro activity of elastase inhibitors correlates with in vivo activity in animal models of emphysema. Thus the novel inhibitors described here should be useful for the treatment of emphysema. Elastase inhibitors have been used orally, by injection or by instillation in the lungs in animal studies (cf. Powers, Am. Rev. Respir. Dis., 127, s54-s58 (1983) and references cited therein). The inhibitors described above can be used by any of these routes. The article by Powers (Am. Rev. Respir. Dis., 127, s54-s58 (1983)) is incorporated herein by reference.

Several other diseases also involve tissue destruction by proteases such as elastase-like and chymotrypsin-like enzymes (cf. Powers, Ad. in Chem., 198, 347-367 (1982)). This article by Powers is incorporated herein by reference. The other diseases include pancreatitis, inflammation, and adult respiratory syndrome. Although correlations between in vitro activity of elastase and chymotrypsin inhibitors and in vivo activity in animal models have not yet be made for these diseases, it is likely that such correlations will be made shortly. And the novel inhibitors can then be used in any cases where such correlations are made.

The following examples are given to illustrate the invention and are not intended to limit it in any manner.

EXAMPLE 1 Preparation of 3-Benzyloxy-4-Chloroisocoumarin

Homophthalic acid (0.5 g, 2.78 mmoles) was placed in benzyl alcohol (5 mL) and 2 drops of concentrated sulfuric acid added. The reaction mixture was heated at 60 to 80 degrees centigrade for 45 min, poured into 50 mL of ice-cold NaHCO₃ (4%) and washed with three portions of ethyl acetate (50 mL). The aqueous layer was acidified to pH 2 with concentrated HCl and allowed to stand overnight. The precipitate was filtered and dried to give benzyl 2-carboxyphenyl acetate (300 mg) as a white solid: R_(f) =0.7 (chloroform/methanol (9/1)). The benzyl 2-carboxyphenyl acetate (300 mg) was dissolved in benzene (7 mL) and PCl₅ (474 mg, 2.28 mmoles) added. The reaction mixture was refluxed for 45 min and the benzene and POCl₃ removed by rotary evaporation. Approximately one-half of the residue was chromatographed on silica gel using benzene as the eluent to give 3-benzyloxy-4-chloroisocoumarin (125 mg) as a pale yellow solid: mp 92 deg centigrade, R_(f) =0.63 (benzene). Anal. Calcd. for C₁₆ H₁₁ O₃ Cl: C, 67.02; H, 3.84. Found: C, 66.83; H, 3.86.

EXAMPLE 2 Preparation of 3-(4-Fluorobenzyloxy)-4-Chloroisocoumarin

Homophthalic acid (4.0 g) was added to a solution containing 4-fluorobenzyl alcohol (10 mL), benzene (10 mL) and H₂ SO₄ (3 drops) and the mixture refluxed at 80 degrees centigrade for 2 h. The reaction mixture was diluted with ethyl acetate (125 mL) and washed with 4% NaHCO₃ (500 mL). The aqueous layer was acidified to pH 3 with concentrated HCl and the solution placed in the refrigerator overnight. The crystals which formed upon cooling were filtered, dissolved in methylene chloride, and dried over magnesium sulfate. The (4-fluorobenzyl) 2-carboxyphenyl acetate was collected from methylene chloride as a white solid (800 mg) and was used without further purification. The (4-fluorobenzyl) 2-carboxyphenyl acetate (0.8 g) was added slowly to a solution of PCl₅ (1.16 g) in benzene (10 mL) and the mixture refluxed at 80 degrees centigrade for 45 min. The benzene was removed, and the residue dissolved in diethyl ether, and filtered. The filtrate was concentrated and collected with isopropyl ether as pale yellow needles (500 mg): mp 127, IR (CH₂ Cl₂) 1743 cm⁻¹. Anal. Calcd. for C₁₆ H₁₀ ClFO₃ : C, 63.07; H, 3.31. Found: C, 62.82; H, 3.36.

EXAMPLE 3 Preparation of 3-Isobutyloxy-4-Chloroisocoumarin

Homophthalic acid (4.0 g) was placed in isobutanol (25 mL) and 3 drops of H₂ SO₄ added. The reaction mixture was heated for 1 h at 100-110 degrees centigrade, poured into 120 mL of ethyl acetate, and washed with 4% NaHCO₃ (15 mL, ×5). The combined aqueous washes were acidified to pH 3 using concentrated HCl and the resulting precipitate collected. The precipitate was dried to give isobutyl 2-carboxyphenyl acetate as a white solid (1.8 g). The isobutyl 2-carboxyphenyl acetate (0.7 g) was placed in a mixture of PCl₅ (1.23 g) and benzene (20 mL) and the mixture heated at reflux for 30 min. The solvent was removed and the residue purified by silica gel chromatography using benzene as the eluent to give product as pale yellow needles (338 mg); mp 48; IR (CH₂ Cl₂) 1740 cm⁻¹. Anal. Calcd. for C₁₃ H₁₃ ClO₃ : C, 61.79; H, 5.19. Found: C, 61.64; H, 5.22.

EXAMPLE 4 Preparation of 7-Nitro-3-Benzyloxy-4-Chloroisocoumarin

4-Nitro-homophthalic acid (5.0 g) was suspended in benzyl alcohol (50 mL) and H₂ SO₄ (3 drops) added. The reaction mixture was heated at 70-80 degrees centigrade for 45 min, diluted into 100 mL of ethyl acetate, and washed with 1 L of 4% NaHCO₃. The aqueous layers were combined, acidified to pH 3 using concentrated HCl, and extracted with 500 mL of ethyl acetate. The ethyl acetate layer was dried over magnesium sulfate and concentrated to give ca. 4 g of crude product, which was titurated with isopropyl ether to give 3.8 grams of benzyl 2-carboxy-4-nitrophenyl acetate: mp. 171-172. Anal. Calcd. for C₁₆ H₁₃ NO₆ : C, 60.96; H, 4.18. Found: C, 60.86; H, 4.18. The benzyl 2-carboxy-4-nitrophenyl acetate (3.0 g) was slowly added to a solution of PCl₅ (4.35 g) in benzene (150 mL) and the reaction mixture stirred overnight at 25 degrees centigrade. The benzene solution was washed with 1.2 L of water, dried over magnesium sulfate and concentrated to give a crude yellow solid. The crude product was titurated with isopropyl ether to give 575 mg of a yellow solid which was purified further by silica gel chromatography using methylene chloride as the eluent. The pure product was crystallized from methylene chloride to give 500 mg of yellow needles: mp 153-154 dec, IR (CH₂ Cl₂) 1785 cm⁻¹, 1622 cm⁻¹. Anal. Calcd. for C₁₆ H₁₀ ClNO₅ : C, 57.94; H, 3.04; N, 4.22. Found: C, 58.04; H, 3.09; N, 4.18.

EXAMPLE 5 Preparation of 4-Chloro-7-Nitro-3-(2-Phenethoxy)isocoumarin

4-Nitrohomophthalic acid (6.0 g, 26.5 mmoles) was suspended in 2-phenylethanol (20 mL), H₂ SO₄ (3 drops) added, and the reaction mixture heated at 120°-130° C. for 3.5 h. The mixture was diluted with ethyl acetate (200 mL) and washed with 4% NaHCO₃ (2×300 mL). Thin layer chromatography indicated that the product was contained primarily in the ethyl acetate layer. After concentration of the ethyl acetate layer, the solid was titurated with isopropyl ether to give the 2-phenylethyl ester of 2-carboxy-4-nitrophenylacetic acid (2.0 g) as a tan solid and was used without further purification. The ester (1.3 g, 3.9 mmoles) was added slowly to a solution of PCl₅ (2.05 g, 9.9 mmoles) in benzene (30 mL) and the mixture refluxed at 80° C. for 1 h. The benzene was removed and the residue titurated with petroleum ether. The crude product was purified by silica gel chromatography using benzene as the eluent to give 4-chloro-7-nitro-3(2-phenethoxy)isocoumarin (0.1 g) as an orange solid: mp 95°-100° C. (dec), one spot on TLC, R_(f) ² =0.5; IR (nujol) 1752 cm⁻¹. Anal. Calcd. for C₁₇ H₁₂ ClNO₅ : C, 59.05; H, 3.50. Found: C, 58.9; H, 3.55.

EXAMPLE 6 Preparation of 7-Amino-4-Chloro-3(2-Phenethoxy)isocoumarin

The 4-chloro-7-nitro-3-(2-phenethoxy)isocoumarin (50 mg, 0.14 mmoles) was dissolved in absolute ethanol (50 mL) and hydrogenated using Pd-C (50 mg) for 2 h. After filtering over celite, the solvent was removed and the residue chromatographed on silica gel using methylene chloride as the eluent to give 7-amino-4-chloro-3(2-phenethoxy)isocoumarin (35 mg) as yellow plates: mp 105°-107° C.; one spot on TLC, R_(f) ³ =0.45; IR (CH₂ Cl₂) 1745 cm⁻¹ ; mass spectrum m/e 315 (M+). Anal. Calcd. for C₁₇ H₁₄ ClNO₃ : C, 64.66; H, 4.47. Found: C, 64.56; H, 4.51.

EXAMPLE 7 Preparation of 7-(N-Tosyl-Phenylalanylamino)-4-Chloro-3-Methoxyisocoumarin

N-Tosyl-phenylalanine acid chloride (77 mg, 0.3 mmoles) and 7-amino-4-chloro-3-methoxyisocoumarin (50 mg, 0.2 mmoles) were suspended in a mixture of methylene chloride/tetrahydrofuran (1:1) and triethylamine (0.037 mL, in 2 mL of methylene chloride) added dropwise with stirring. After stirring at 25° C. for 2 h, the reaction solvents were removed and the residue dissolved in ethyl acetate. After washing with 10% citric acid (3×30 mL) and 4% NaHCO₃ (2×30 mL), the ethyl acetate was dried over magnesium sulfate and concentrated. The crude product was purified by silica gel chromatography using a 1% mixture of methanol in methylene chloride and the 7-(N-tosyl-phenylalanylamino)-4-chloro-3-methoxyisocoumarin (22 mg) collected from methanol/isopropyl ether as a pale yellow solid: mp 222°-224° C. (dec); one spot on TLC, R_(f) ³ =0.3; IR (nujol) 1750 cm⁻¹ ; mass spectrum m/e 527 (M+1). Anal. Calcd. for C₂₆ H₂₃ ClN₂ O₆ S 1/2 H₂ O: C, 58.26; H, 4.53. Found: C, 58.28; H, 4.50.

EXAMPLE 8 Preparation of 3-Propyl-1H-2-Benzopyran-1,4(3H)-dione

A solution of 3-bromo,3-(1-bromobutyl)-1(3H)-isobenzofuranone (500 mg) in Me₂ SO (15 mL) was added dropwise to a solution of 0.1M Hepes buffer (pH 7.5, 50 mL) containing 0.5M NaCl and Me₂ SO (35 mL). The mixture was stirred at 25 degrees centigrade, diluted with water (100 mL) and extracted with ethyl acetate (50 mL, ×2). The extracts were combined, washed with water (50 mL,×5), dried over magnesium sulfate, and concentrated under reduced pressure. The residue was chromatographed on silica gel using benzene as the eluent to give the product as a white solid (170 mg): mp 44-45 degrees centigrade; IR (nujol), 1730, 1700 cm⁻¹. Anal. Calcd. for C₁₂ H₁₂ O₃ : C, 70.57; H, 5.92. Found: C, 70.55; H, 5.96.

Other illustrative examples are given in references cited below: 3-chloroisocoumarin: Davies and Poole, J. Chem. Soc., pp 1616-1620 (1928); 3,4-dichloroisocoumarin: Milevskaya, Belinskaya, and Yagupol'skii, Zhur. Org. Khim. 9, pp 2145-2149, (1973); 3-methoxy-4-chloroisocoumarin and 3-ethoxy-4-chloroisocoumarin: Tirodkar and Usgaonkar, Ind. J. Chem. 7, pp 1114-1116, (1969); 7-nitro-3-methoxy-4-chloroisocoumarin and 7-amino-3-methoxy-4-chloroisocoumarin: Choksey and Usgaonkar, Ind. J. Chem. 14B, pp 596-598, (1976). 

What is claimed is:
 1. A method of treating emphysema in animals by inhibiting in vivo the enzymatic activity of serine proteases, comprising the step of administering to said animal that amount of a compound effective to inhibit said activity having the following structure: ##STR3## wherein X is selected from the group consisting of O, and S,Z is selected from the group consisting of H, halogen, C₁₋₆ alkyl, C₁₋₆ alkyl with an attached phenyl, C₁₋₆ fluorinated alkyl, C₁₋₆ alkoxy, C₁₋₆ fluorinated alkoxy, C₁₋₆ alkoxy with an attached phenyl, benzyloxy, 4-fluorobenzyloxy, --OCH₂ C₆ H₄ --R' (2-substituent), --OCH₂ C₆ H₄ --R' (3-substituent), --OCH₂ C₆ H₄ --R' (4-substituent), --OCH₂ C₆ H₃ --R₂ ' (2,3-substituents), --OCH₂ C₆ H₃ --R₂ ' (2,4-substituents), --OCH₂ C₆ H₃ --R₂ ' (2,5-substituents), --OCH₂ C₆ H₃ --R₂ ' (2,6-substituents), --OCH₂ C₆ H₃ --R₂ ' (3,4-substituents), and --OCH₂ C₆ H₃ --R₂ ' (3,5-substituents), R' is selected from the group consisting of H, halogen, trifluoromethyl, NO₂, cyano, methyl, methoxy, acetyl, carboxyl, OH, and amino, Y is selected from the group consisting of H, halogen, trifluoromethyl, methyl, OH, and methoxy, R is selected from the group consisting of H, OH, NH₂, NO₂, halogen, --NH--C(NH)--NH₂, --C(NH)NH₂, C₁₋₆ alkoxy, C₁₋₆ fluorinated alkoxy, C₁₋₆ alkyl, C₁₋₆ alkylamino, M-AA, M-AA-NH, and M-NH, wherein AA is selected from the group consisting of alanine, valine, leucine, isoluecine, proline, methionine, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, beta-alanine, norleucine, norvaline, alpha-aminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, and sarcosine, wherein M is selected from the group consisting of hydrogen, lower alkanoyl having 1 to 6 carbons, carboxylalkanyl, hydroxyalkanoyl, amino-alkanoyl, benzene sulfonyl, tosyl, benzoyl, and lower alkyl sulfonyl having 1 to 6 carbons.
 2. A method of treating emphysema in animals by inhibiting in vivo the enzymatic activity of serine proteases, comprising the step of administering to said animal that amount of a compound effective to inhibit said activity having the following structure: ##STR4## wherein R₁ is selected from the group consisting of H, halogen C₁₋₆ alkyl, C₁₋₆ alkyl with an attached phenyl, C₁₋₆ fluorinated alkyl, C₁₋₆ alkoxy, C₁₋₆ fluorinated alkoxy, C₁₋₆ alkoxy with an attached phenyl, benzyloxy, 4-fluorobenzyloxy, --OCH₂ C₆ H₄ --R' (2-substituent), --OCH₂ C₆ H₄ --R' (3-substituent), --OCH₂ C₆ H₄ --R' (4-substituent), --OCH₂ C₆ H₃ --R₂ ' (2,3-substituents), --OCH₂ C₆ H₃ --R₂ ' (2,4-substituents), --OCH₂ C₆ H₃ --R₂ ' 2,5-substituents), --OCH₂ C₆ H₃ --R₂ ' (2,6-substituents), --OCH₂ C₆ H₃ --R₂ ' (3,4-substituents), and --OCH₂ C₆ H₃ --R₂ ' (3,5-substituents),R' is selected form the group consisting of H, halogen, trifluoromethyl, NO₂, cyano, methyl, methoxy, acetyl, carboxyl, OH, and amino, and R₂ is selected from the group consisting of H, OH, NH₂, NO₂, halogen, --NH--C(NH)--NH₂, --C(NH)NH₂, C₁₋₆ alkoxy, C₁₋₆ fluorinated alkoxy, C₁₋₆ alkyl, and C₁₋₆ alkylamino.
 3. A process for the inhibition of the enzymatic activity of serine proteases comprising the step of adding to a medium containing the protease that amount of inhibitor effective to inhibit said activity having the following structure: ##STR5## wherein X is selected from the group consisting of O, and S,Z is selected from the group consisting of H, halogen, C₁₋₆ alkyl, C₁₋₆ alkyl with an attached phenyl, C₁₋₆ fluorinated alkyl, C₁₋₆ alkoxy, C₁₋₆ fluorinated alkoxy, C₁₋₆ alkoxy with an attached phenyl, benzyloxy, 4-fluorobenzyloxy, --OCH₂ C₆ H₄ --R' (2-substituent), --OCH₂ C₆ H₄ --R' (3-substituent), --OCH₂ C₆ H₄ --R' (4-substituent), --OCH₂ C₆ H₃ --R₂ ' (2,3-substituents), --OCH₂ C₆ H₃ --R₂ ' (2,4-substituents), --OCH₂ C₆ H₃ --R₂ ' (2,5-substituents), --OCH₂ C₆ H₃ --R₂ ' (2,6-substituents), --OCH₂ C₆ H₃ --R₂ ' (3,4-substituents), and --OCH₂ C₆ H₃ --R₂ ' (3,5-substituents), R' is selected from the group consisting of H, halogen, trifluoromethyl, NO₂, cyano, methyl, methoxy, acetyl, carboxyl, OH, and amino, Y is selected from the group consisting of H, halogen, trifluoromethyl, methyl, OH, and methoxy, R is M-AA-NH-, wherein AA is selected from the group consisting of alanine, valine, leucine, isoleucine, proline, methionine, phenylalanine, tryptophan, glycine, serine, threonine, cysteine, tyrosine, asparagine, glutamine, aspartic acid, glutamic acid, lysine, arginine, histidine, beta-alanine, norleucine, norvaline, alphaaminobutyric acid, epsilon-aminocaproic acid, citrulline, hydroxyproline, ornithine, and sarcosine, wherein M is selected from the group consisting of hydrogen, lower alkanoyl having 1 to 6 carbons, carboxylalkanoyl, hydroxyalkanoyl, amino-alkanoyl, benzene sulfonyl, tosyl, benzoyl, and lower alkyl sulfonyl having 1 to 6 carbons. 